Colorimetric assays of various fluids for the determination of chemical or biological substances (identified as analytes herein) are well known. Such assays are particularly important in clinical chemistry as the medical and veterinary professions attempt to rapidly and economically diagnose and treat ailments in humans and animals. As a result, researchers are continually searching for more sensitive and less expensive means for doing such assays.
A relatively recent contribution to the clinical chemistry art was the development of thin-film multilayer analytical elements such as those described, for example, in U.S. Pat. Nos. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al) and 4,144,306 (issued Mar. 13, 1979 to Figueras). Those elements are generally described as having a porous spreading layer, a reagent layer and a registration layer carried on a nonporous support. The support may be designed to transmit all or part of incident radiation in order to facilitate measurement of detectable species at particular wavelengths.
Another significant advance in the art is described in U.S. Pat. No. 4,089,747 (issued May 16, 1978 to Bruschi). This reference describes certain triarylimidiazole leuco dyes which have become very useful in assays for hydrogen peroxide or glucose, uric acid and other analytes where hydrogen peroxide is generated as a result of the presence of the analyte.
The determination of the activity of creatine kinase (abbreviated herein to CK, but also known at creatine phosphokinase, CPK, or ATP:creatine phosphotransferase E.C.2.7.3.2.) in human serum is considered one of the most sensitive laboratory methods for diagnosing diseases of skeletal muscles and diseases of the myocardium. CK determinations are useful, for example, for diagnosis of progressive muscular dystrophy, dermatomyositis and especially myocardial infarctions. Determination of CK-MB, one of the three isoenzymes of CK, is important for the evaluation of the damage to the heart in the case of cardiac infarctions.
Most standard assays for a number of analytes, including creatine kinase, generally measure a change in ultraviolet light absorption. Light incident on the test sample can be either broad band radiation or filtered radiation, depending upon the optical equipment and procedure used.
It has been observed, however, that some compounds used in such assays are photosensitive, i.e. they change or promote changes prematurely in response to light. In particular, some dyes or dye precursors useful in assays (e.g. the triarylimidazole leuco dyes described above) exhibit undesirable photosensitivity in various assays, including assays for CK or other enzymes. As a result, the dyes or their precursors prematurely provide an unwanted optical density change and a high rate of background formation in the assay. In other words, there is an unwanted detectable change in rate. This problem was not recognized in assays of analytes which are present in high concentrations because the response from the analyte is so much greater than the unwanted background. However, the problem became pronounced in instances where the analyte is present in relatively low concentrations. A high rate of background formation significantly reduces assay sensitivity and precision.
While many photosensitive dyes or dye precursors are useful for assays of low level analytes, their use may be restricted due to their photosensitivity. Hence, it would be desirable to have a means for using such dyes in a highly sensitive assay without concern for their photosensitivity.